M1-type polarized macrophage contributes to brain damage through CXCR3.2/CXCL11 pathways after RGNNV infection in grouper.

The vertebrate central nervous system (CNS) is the most complex system of the body. The CNS, especially the brain, is generally regarded as immune-privileged. However, the specialized immune strategies in the brain and how immune cells, specifically macrophages in the brain, respond to virus invasion remain poorly understood. Therefore, this study aimed to examine the potential immune response of macrophages in the brain of orange-spotted groupers (Epinephelus coioides) following red-spotted grouper nervous necrosis virus (RGNNV) infection. We observed that RGNNV induced macrophages to produce an inflammatory response in the brain of orange-spotted grouper, and the macrophages exhibited M1-type polarization after RGNNV infection. In addition, we found RGNNV-induced macrophage M1 polarization via the CXCR3.2- CXCL11 pathway. Furthermore, we observed that RGNNV triggered M1 polarization in macrophages, resulting in substantial proinflammatory cytokine production and subsequent damage to brain tissue. These findings reveal a unique mechanism for brain macrophage polarization, emphasizing their role in contributing to nervous tissue damage following viral infection in the CNS.

Slc43a2+ T cell metastasis from spleen to brain in RGNNV infected teleost.

The origin of T cells in the teleost's brain is unclear. While viewing the central nervous system (CNS) as immune privileged has been widely accepted, previous studies suggest that T cells residing in the thymus but not in the spleen of the teleost play an essential role in communicating with the peripheral organs. Here, we identified nine T cell subpopulations in the thymus and spleen of orange-spotted grouper (Epinephelus coioices) through single-cell RNA-sequencing analysis. After viral CNS infection with red-spotted grouper nervous necrosis virus (RGNNV), the number of slc43a2+ T cells synchronously increased in the spleen and brain. During the infection tests in asplenic zebrafish (tlx1▲ zebrafish model), no increase in the number of slc43a2+ T cells was observed in the brain. Single-cell transcriptomic analysis indicated that slc43a2+ T cells mature and functionally differentiate within the spleen and then migrate into the brain to trigger an immune response. This study suggests a novel route for T cell migration from the spleen to the brain during viral infection in fish.

Effects of Dietary Protein Levels on Growth, Digestive Enzyme Activity, Antioxidant Capacity, and Gene Expression Related to Muscle Growth and Protein Synthesis of Juvenile Greasyback Shrimp (Metapenaeus ensis).

An 8-week feeding trial was conducted to assess the effects on growth, antioxidant capacity, digestive enzyme activity, and gene expression related to muscle growth and protein synthesis of juvenile greasyback shrimp (Metapenaeus ensis) using five experimental diets containing 29.37%, 34.30%, 39.11%, 44.05%, and 49.32% of protein. The results demonstrated that juvenile greasyback shrimp consuming 39.11%, 44.05%, and 49.32% dietary protein had a significantly higher final body weight (FBW), weight gain (WG), feed conversion ratio (FCR), and specific growth rate (SGR) than other groups (p < 0.05). The protein efficiency ratio (PER) showed a significantly quadratic pattern with increasing dietary protein levels (p < 0.05). The highest trypsin and pepsin activities were observed in the group with a protein level of 44.05% (p < 0.05). Relatively higher superoxide dismutase (SOD) activity was found in groups with protein levels of 39.11% (p < 0.05). Alkaline phosphatase (AKP) and catalase (CAT) activity showed a significantly linear increasing pattern with increasing protein intake up to 44.05%, and then decreased gradually (p < 0.05). Compared to the dietary 29.37% protein level, the expression levels of myogenic regulatory factors (mef2α, mlc, and myf5) and mTOR pathway (mtor, s6k, akt, and pi3k)-related genes were significantly up-regulated in muscle with 39.11%, 44.05%, and 49.32% dietary protein levels (p < 0.05). The AAR pathway (gcn2, eif2α, and atf4)-related gene expression levels were significantly lower in muscles with 39.11%, 44.05%, and 49.32% protein levels than in other groups (p < 0.05). Based on the broken-line regression analysis of SGR, the estimated appropriate dietary protein requirement for juvenile greasyback shrimp is 38.59%.

The complete mitochondrial genome of Allogalathea elegans (Adams & White, 1848) (Decapoda: Galatheidae).

The genus Allogalathea belongs to the subfamily Galatheoidea of the family Galatheidae. Here, we report a mitogenome of Allogalathea elegans (Adams & White, 1848). In this study, we obtained the complete mitochondrial genome of Allogalathea elegans by sequencing, which was 16,263 bp in length. The mitogenome contained 37 genes, including the typical set of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 Ribosomal RNA (rRNA) genes. The nucleotides A, C, G, and T distribution was 36.40%, 19.44%, 9.09%, and 35.07%, respectively. The length of the total protein-coding genes was 11,172 bp, which accounts for 68.69% of the whole mitochondrial genome. The phylogenetic result generated by IQ-Tree based on 13 PGCs showed that the infraorder Anomura is monophyletic, and the infraorder Anomura is a sister group of the infraorder Glypheidea. The discovery of the complete mitochondrial genome of A. elegans would help to conduct in-depth research on the infraorder Anomura.

The complete mitochondrial genome of Hypseleotris cyprinoides (Perciformes: eleotridae).

In this study, we obtained the complete mitochondrial genome of Hypseleotris cyprinoides, which was 16520 bp in length. The mitogenome contained 37 genes, including the typical set of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 Ribosomal RNA (rRNA) genes. A, C, G, and T distribution was 28.57%, 29.91%, 16.99%, and 24.53%, respectively. The length of the total protein-coding genes was 11441 bp, which accounts for 66.80% of the whole mitochondrial genome. The Maximum Likelihood (ML) phylogenetic analysis based on the concatenated nucleotide sequences of 13 PCGs showed that H.cyprinoides as a sister species to Hypseleotris klunzingeri was clustered in the family Hypseleotris. The discovery of the complete mitochondrial genome of H.cyprinoides would help to conduct in-depth research on Hypseleotris.

The Expression Pattern of Insulin-Like Growth Factor Subtype 3 (igf3) in the Orange-Spotted Grouper Epinephelus coioides and Its Function on Ovary Maturation.

A new insulin-like growth factor (Igf) subtype 3 (igf3) has recently been found in the bony fish orange-spotted grouper (Epinephelus coioides). However, the role of igf3 in the maturation of the ovary and sex differentiation in E. coioides is currently unknown. We examined the ovarian localization and receptor binding of the novel ortholog Igf3 using qRT-PCR, and Western blotting, combined with in situ hybridization and immunohistochemistry methods. Results demonstrated the presence of igf3 mRNA and protein in mature oocytes. Furthermore, Igf3 protein expression was not detected in testis, brain, kidney and liver homogenates. The calculated molecular weight of Igf3 was 22 kDa, which was consistent with the deduced amino acid sequence from the full-length open reading frame. The immunoreactivity showed that Igf3 was strongly present in the follicle staining fully-grown stage. The igf3 mRNA expression level was significantly positively correlated with ovarian follicular maturation. Meanwhile, Igf3 increased germinal-vesicle breakdown in a time- and dose-dependent manner. In vitro, treatment of primary ovarian cells with Igf3 up-regulated significantly the mRNA expression level of genes related to sex determination and reproduction such as forkhead boxl2 (foxl2), dosage-sensitive sex reversal adrenal hypoplasia critical region on chromosome x gene 1 (dax1), cytochrome P450 family 19 subfamily member 1 a (cyp19a1a), cytochrome P450 family 11 subfamily a member 1 a (cyp11a1a) and luteinizing hormone receptor 1 (lhr1). Overall, our results demonstrated that igf3 promotes the maturation of the ovary and plays an important role in sex differentiation in E. coioides.

Effects of nanoplastic exposure on the immunity and metabolism of red crayfish (Cherax quadricarinatus) based on high-throughput sequencing.

Previous studies have shown that nanoplastics (NPs) are harmful pollutants that threaten aquatic organisms and ecosystems, however, less research has been conducted on the hazards of NPs for aquaculture animals. In this study, Cherax quadricarinatus was used as an experimental model to evaluate the possible effects of three concentrations (25, 250 and 2500 µg/L) of NPs on red crayfish. The toxicological effects of NPs on this species were investigated based on transcriptomics and microbiome. A total of 67,668 genes were obtained from the transcriptome. The annotation rate of the four major libraries (Nr, KEGG, KOG, Swissprot) was 40.17 %, and the functions of differential genes were mainly related to antioxidant activity, metabolism and immune processes. During the experiment, the activities of superoxide dismutase (SOD) and catalase (CAT) in the high concentration group were significantly decreased, while the concentration of malondialdehyde (MDA) increased after nanoplastics (NPs) exposure, and SOD1, Jafrac1 were significantly reduced at high concentrations. expression is inhibited. The immune genes LYZ and PPO2 were highly expressed at low concentrations and suppressed at high concentrations. After 14 days of exposure to NPs, significant changes in gut microbiota were observed, such as decreased abundances of Actinobacteria, Bacteroidetes, and Firmicutes. NPs compromise host health by inducing changes in microbial communities and the production of beneficial bacterial metabolites. Overall, these results suggest that NPs affect immune-related gene expression and antioxidant enzyme activity in red crayfish and cause redox imbalance in the body, altering the composition and diversity of the gut microbiota.

Enhanced Muscle Fibers of Epinephelus coioides by Myostatin Autologous Nucleic Acid Vaccine.

Epinephelus coioides is a fish species with high economic value due to its delicious meat, high protein content, and rich fatty acid nutrition. It has become a high-economic fish in southern parts of China and some other Southeast Asian countries. In this study, the myostatin nucleic acid vaccine was constructed and used to immunize E. coioides. The results from body length and weight measurements indicated the myostatin nucleic acid vaccine promoted E. coioides growth performance by increasing muscle fiber size. The results from RT-qPCR analysis showed that myostatin nucleic acid vaccine upregulated the expression of myod, myog and p21 mRNA, downregulated the expression of smad3 and mrf4 mRNA. This preliminary study is the first report that explored the role of myostatin in E. coioides and showed positive effects of autologous nucleic acid vaccine on the muscle growth of E. coioides. Further experiments with increased numbers of animals and different doses are needed for its application to E. coiodes aquaculture production.

Liposome-Encapsulated Rec8 and Dmrt1 Plasmids Induce Red-Spotted Grouper (Epinephelus akaara) Testis Maturation.

In fish, the maturity of gonads plays an important role in the development and reproduction of the population, and it also dictates the success of captive breeding. Therefore, finding ways to promote gonadal maturation is an important goal in aquaculture. In this study, we injected recombinant dmrt1 and rec8 overexpression plasmids packaged in liposomes into the immature testis of red-spotted grouper (Epinephelus akaara) and measured the expression of Dmrt1 and Rec8 protein in vivo. Gonadosomatic index (GSI) and gonadal histology analyses showed that the testis developed from the immature to the mature state within 7 days after plasmid injection. Additionally, the spermatozoa concentration and motility in plasmid-injected fish was the same as that of naturally mature fish. These results provided evidence that delivery of dmrt1 and rec8 expression plasmids into the testis via injection induced testis maturation in vivo.

Polystyrene nanoplastics alter virus replication in orange-spotted grouper (Epinephelus coioides) spleen and brain tissues and spleen cells.

Polystyrene nanoplastics (PS-NPs) are known to impair the function of the digestive system, intestinal flora, immune system, and nervous system of marine organisms. We tested whether PS-NPs influence viral infection of orange-spotted grouper (Epinephelus coioides). We found that grouper spleen (GS) cells took up PS-NPs at exposure concentrations of 5, 50, and 500 µg/mL and experienced cytotoxicity at 50 and 500 µg/mL concentrations. At 12 h after exposure to 50 µg/mL of PS-NPs, the replication of Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) increased in GS cells after their invasion. Juvenile fish exposed to 300 and 3000 µg/L of PS-NPs for 7 d showed PS-NPs uptake to the spleen and vacuole formation in brain tissue. Moreover, PS-NPs exposure accelerated SGIV replication in the spleen and RGNNV replication in the brain. PS-NP exposure also decreased the expression of toll-like receptor genes and interferon-related genes before and after virus invasion in vitro and in vivo, thus reducing the resistance of cells and tissues to viral replication. This is the first report that PS-NPs have toxic effects on GS cells and spleen and brain tissues, and it provides new insights into assessing the impact of PS-NPs on marine fish.

Efficient RNA Virus Targeting via CRISPR/CasRx in Fish.

The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. IMPORTANCE RNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.

Single-cell RNA-seq landscape midbrain cell responses to red spotted grouper nervous necrosis virus infection.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

The ERß-CXCL19/CXCR4-NFκB pathway is critical in mediating the E2-induced inflammation response in the orange-spotted grouper (Epinephelus coioides).

The main physiological function of 17ß-estradiol (E2) in vertebrates is to regulate sexual development and reproduction. In fish, especially hermaphroditic fish, estrogen is often used to aid reproduction, but it also can trigger an inflammatory response. However, the molecular mechanism for this E2-induced inflammatory reaction is not clear. In this study, we found that the ERß-CXCL19/CXCR4-NFκB cascade regulated the E2-induced inflammatory response in the orange-spotted grouper (Epinephelus coioides). Strikingly, E2 treatment resulted in significantly high expression of inflammatory cytokines and induced phosphorylation and degradation of IκBα and translocation of NFκB subunit p65 to the nucleus in grouper spleen cells. However, the E2-induced inflammatory response could be prevented by the broad estrogen receptor (ER) ligand ICI 182,780. Moreover, the luciferase assay showed that E2 induced the inflammatory response by activating the promotor of chemokine CXCL19 through ERß1 and ERß2. Knockdown of CXCL19 blocked the E2-induced inflammatory response and NFκB nucleus translocation. Additionally, knockdown of chemokines CXCR4a and CXCR4b together, but not alone, blocked the E2-induced inflammatory response. The immunofluorescence assay and co-immunoprecipitation analysis showed that CXCL19 mediated the E2-induced inflammatory response by activating CXCR4a or CXCR4b. Taken together, these results showed that the ERß-CXCL19/CXCR4-NFκB pathway mediated the E2-induced inflammatory response in grouper. These findings are valuable for future comparative immunological studies and provide a theoretical basis for mitigating the adverse reactions that occur when using E2 to help fish reproduce.

Cloning and characterization of rec8 gene in orange-spotted grouper (Epinephelus coioides) and Dmrt1 regulation of rec8 promoter activity.

Meiosis is a specialized type of cell division critical for gamete production during sexual reproduction in eukaryotes. The meiotic recombination protein Rec8 has been identified as an important factor in germ cell meiotic initiation in vertebrates; however, its equivalent role in teleosts is poorly characterized. In this study, we cloned and sequenced the rec8 gene from orange-spotted grouper (Epinephelus coioides). The cDNA sequence consisted of 2244 base pairs (bp), including a 5' untranslated region (UTR) of 198 bp and a 3'UTR of 284 bp. The open reading frame of grouper rec8 was 1752 bp, encoding 584 amino acids. Expression levels of rec8 were higher in the ovary, intersex gonad, and testis. A neighbor-joining phylogenetic tree based on the deduced amino acid sequence indicated a common origin for grouper and other teleost rec8 molecules. Immunohistochemistry using a polyclonal anti-Rec8 antibody localized the protein in the oogonia and primary oocytes in the ovary and in spermatogonia and spermatocytes in the intersex gonad and testis, suggesting that Rec8 may play an important role in the meiotic division and the development of grouper germ cells. In addition, we found that the transcription factor Dmrt1 increased rec8 promoter activity through the second binding site, based on dual-luciferase assays. Together, these results suggest that Rec8 plays a crucial role in meiosis and may be regulated by Dmrt1 to affect meiosis in groupers.

Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): gonadal restructuring, sex hormone shifts and gene profiles.

Sexual patterns of teleosts are extremely diverse and include both gonochorism and hermaphroditism. As a protogynous hermaphroditic fish, all orange-spotted groupers (Epinephelus coioides) develop directly into females, and some individuals change sex to become functional males later in life. This study investigated gonadal restructuring, shifts in sex hormone levels and gene profiles of cultured mature female groupers during the first (main) breeding season of 2019 in Huizhou, China (22° 42' 02.6″ N, 114° 32' 10.1″ E). Analysis of gonadal restructuring revealed that females with pre-vitellogenic ovaries underwent vitellogenesis, spawning and regression and then returned to the pre-vitellogenic stage in the late breeding season, at which point some changed sex to become males via the intersex gonad stage. A significant decrease in the level of serum 17ß-estradiol (E2) was observed during ovary regression but not during sex change, whereas serum 11-ketotestosterone (11-KT) concentrations increased significantly during sex change with the highest concentration in newly developed males. Consistent with serum hormone changes, a significant decrease in cyp19a1a expression was observed during ovary regression but not during sex change, whereas the expression of cyp11c1 and hsd11b2 increased significantly during sex change. Interestingly, hsd11b2 but not cyp11c1 was significantly upregulated from the pre-vitellogenic ovary stage to the early intersex gonad stage. These results suggest that a decrease in serum E2 concentration and downregulation of cyp19a1a expression are not necessary to trigger the female-to-male transformation, whereas increased 11-KT concentration and upregulation of hsd11b2 expression may be key events for the initiation of sex change in the orange-spotted grouper.

In vitro effects of androgen on testicular development by the AR-foxl3-rec8/fbxo47 axis in orange-spotted grouper (Epinephelus coioides).

In orange-spotted grouper, androgen can promote the development of testis and spermatogenesis, but the effect of androgen on testis development is unclear. Forkhead box L 3 (Foxl3) is important in the development of fish testis. Rec8 and fbxo47 are involved in meiosis, which impacts spermatogenesis. The present study investigated the plausible role of testis development through the Foxl3 transcriptional regulation of rec8 and fbxo47. The results of tissue distribution showed that rec8 and fbxo47 are highly expressed in gonad. In addition, the highest expression of foxl3, rec8, and fbxo47 was in the testis and intersex compared with the other stages of gonadal development, suggesting that foxl3, rec8, and fbxo47 are important in testis development. In addition, by using dual-luciferase assays, we found that the androgen can increase foxl3 promoter activity and Foxl3 can upregulate rec8 and fbxo47 promoter activity. Furthermore, the addition of ß-testosterone significantly increased foxl3, rec8, and fbxo47 promoter activity. Together, these results suggest that foxl3 plays a decisive role in testis development by regulating the expression of rec8 or fbxo47 and imply that AR-foxl3-rec8/fbxo47 affects the testis development pathway.

Retinoic acid and androgen influence germ cells development and meiotic initiation in juvenile orange-spotted grouper, Epinephelus coioides.

Meiosis is essential for germ cells development for all sexually reproducing species. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in mammals, birds and tetrapods. Here, we investigated the effects of RA on meiotic initiation and sex determination in protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Expression profile investigations of meiotic marker genes during gonadal development indicated that germ cells undergone meiosis approximately at 180 days after hatching in the orange-spotted grouper. RA synthase inhibitor treatments on juvenile orange-spotted groupers resulted in impeded germ cells development and delayed meiotic initiation with simultaneous down-regulation of vasa, dazl, sycp3 and rec8, which was rescued by exogenous RA administration. Additionally, exogenous androgen treated fish showed a delayed meiotic initiation consistent with decreased sycp3 and rec8 expression and were directed to a spermiogenesis fate. Our results imply that meiotic initiation in the orange-spotted grouper is strongly influenced by RA and androgen, and the regulation of meiotic initiation may involve in the spermatogenesis induced by exogenous androgen.

Effect of starvation and refeeding on growth, gut microbiota and non-specific immunity in hybrid grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂).

Environmental changes can lead to food deprivation among aquatic animals. The main objective of this present research was to assess the effect of starvation and refeeding on growth, gut microbiota and non-specific immunity in a hybrid grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂). A total of 120 fish with an average weight of 74.16 ± 12.08 g were randomly divided into two groups (control group and fasted-refed group). The control group was fed until satiation for 60 days, while the fasted-refed group was fasted for 30 days and then fed to satiation for 30 days. The results showed that starvation led to a significantly decreased growth performance parameters [weight gain rate (WGR) and specific weight gain rate (SGR), while the feeding rate (FR) ] increased during the refeeding, non-specific immunity was significantly improved (p < 0.05) during the first 15 days of starvation, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), lysozyme (LYM) and catalase (CAT). However, non-specific immunity decreased at 30 days of starvation, the expression of genes related to immunity, such as TNF-α, was upregulated (p < 0.05) during starvation, while the expression levels of IL-17 and IFN-γ was reduced (p < 0.05). The expression of IFN-γ and IL-1ß peaked during refeeding. Starvation led to significantly decreased abundance and diversity of intestinal microflora, with a higher abundance of Vibrio and a lower abundance of Brevibacillus, Bifidobacterium, Alloprevotella in the fasted-refed group during refeeding than in the control group. The above results reveal that starvation stimulates changes in growth, non-specific immunity, and the gut microbiota, providing new insights for the study of fish habitat selection and adaptability to environmental changes.

Assessment of the Effects of Bisphenol A on Dopamine Synthesis and Blood Vessels in the Goldfish Brain.

Bisphenol A (BPA) is an abundant contaminant found in aquatic environments. While a large number of toxicological studies have investigated the effects of BPA, the potential effects of BPA exposure on fish brain have rarely been studied. To understand how BPA impacts goldfish brains, we performed a transcriptome analysis of goldfish brains that had been exposed to 50 µg L-1 and 0 µg L-1 BPA for 30 days. In the analysis of unigene expression profiles, 327 unigenes were found to be upregulated and 153 unigenes were found to be downregulated in the BPA exposure group compared to the control group. Dopaminergic signaling pathway-related genes were significantly downregulated in the BPA exposure group. Furthermore, we found that serum dopamine concentrations decreased and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) staining was present in dopamine neurons enriched regions in the brain after BPA exposure, suggesting that BPA may disrupt dopaminergic processes. A KEGG analysis revealed that genes involved in the fluid shear stress and atherosclerosis pathway were highly significantly enriched. In addition, the qRT-PCR results for fluid shear stress and atherosclerosis pathway-related genes and the vascular histology of the brain showed that BPA exposure could damage blood vessels and induce brain atherosclerosis. The results of this work provide insights into the biological effects of BPA on dopamine synthesis and blood vessels in goldfish brain and could lay a foundation for future BPA neurotoxicity studies.

Toxic effects of bisphenol A on goldfish gonad development and the possible pathway of BPA disturbance in female and male fish reproduction.

Bisphenol A (BPA) is an abundant endocrine-disrupting compound that is found in the aquatic environment and has adverse effects on fish reproduction; however, the exact pathway of these impacts is unclear. In this study, the different effects of BPA on ovarian and testis development in goldfish (Carassius auratus) and the different mechanisms underlying these effects were investigated. The gonadosomatic index (GSI) and gonadal histology demonstrated that BPA diminished ovarian maturation in goldfish, which recovered after BPA treatment withdrawal. In males, BPA disrupted testis maturation, but this disruption could not be recovered after BPA treatment withdrawal. The hypothalamic-pituitary-gonad (HPG) axis-related genes sgnrh, fshß and lhß were significantly decreased in BPA-treated female fish, while no changes in sex steroid hormone levels and no TUNEL and PCNA staining were found in the ovary, suggesting that BPA may reduce ovarian maturation through the HPG axis. In male fish, TUNEL staining was found in 1 µg L-1 BPA-exposed germ cells and 50 and 500 µg L-1 BPA-exposed Leydig cells. Decreases in 11-KT levels were also found in 50 and 500 µg L-1 BPA-exposed fish, but BPA did not affect genes associated with the HPG axes. This result shows that BPA disrupts testis maturation through apoptosis of germ cells and Leydig cells, thus inducing decreases in 11-KT levels that disrupt spermatogenesis. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying BPA disturbance of goldfish reproduction.